PROJECT SUMMARY/ABSTRACT We can enhance antibody therapy by stimulating the action of natural killer (NK) cells by a single second antibody. We can trigger a global therapeutic immune response by ?in situ vaccination?, the injection of TLR ligands directly into one tumor site. We are conducting clinical trials of both of these approaches and the early results are very promising. The addition of antibodies that block immune checkpoints make these approaches even more powerful. We will conduct preclinical and clinical projects based on these platforms. 1. Enhancing antibody therapy of cancer with a second antibody that stimulates activated NK cells. We discovered that the therapeutic activity of Rituximab and other mAbs (Traztuzumab and Cetuximab) are enhanced by the sequential addition of a second antibody against CD137 (41BB), an activation molecule on the NK killer cells. We are now leading clinical trials that test the addition of CD137 antibodies to Rituximab in patients with B cell lymphoma. Simultaneously, we are testing bispecific antibodies that bind both to the tumor and to activation markers on NK cells. 2. Therapeutic In Situ Vaccination. We demonstrated that a global therapeutic anti-tumor immune response can be triggered by the injection of TLR ligands into a single tumor site. We are conducting a phase I/II clinical trial in lymphoma patients, testing the combination of low dose XRT followed by intra-tumoral injection of both a TLR agonist and a low dose of anti CTLA4 antibody. We now have preclinical evidence for synergy between in situ vaccination and Ibrutinib, an inhibitor of BTK and ITK, a combination that works also in solid tumors. 3. The Immune Response against immunoglobulin (Ig)-derived peptides in B cell lymphoma B-cell lymphomas express a unique immunoglobulin idiotype that distinguishes malignant cells from normal B- cells. Idiotypes are ideal targets for immunotherapy because they are expressed on all the malignant cells in the patient. We discovered that patients have T cells that recognize peptides derived from idiotypes that are displayed in MHC class II molecules. Now we will design new immunotherapy directed at MHC II-idiotypes. 4. New Methods of Immune Monitoring A critical component of each of our immunotherapy projects is an intense focus on monitoring cells in the tumor microenvironment. We will obtain samples from the same tumor sites: before, during, and after the experimental therapies and analyze the cell populations by high-dimensional flow cytometry. These data sets will be compared across trials that test different modalities of immunotherapy. We will detect activated T cells in patients by novel non-invasive imaging techniques to track the effects of immunotherapies in real time .We will detect the tumor-reactive cell populations in tissue sections by Multiplexed Ion Beam Imaging (MIBI). This system can interrogate 10 or more parameters per cell with high spatial resolution on sections from FFPE embedded tissues.